We examined the effect of short-and long-term changes in temperature on gene expression, protein abundance, and the activity of the alternative oxidase and cytochrome oxidase pathways (AOP and COP, respectively) in Arabidopsis thaliana. The AOP was more sensitive to short-term changes in temperature than the COP, with partitioning to the AOP decreasing significantly below a threshold temperature of 20°C. AOP activity was increased in leaves, which had been shifted to the cold for several days, but this response was transient, with AOP activity subsiding (and COP activity increasing) following the development of leaves in the cold. The transient increase in AOP activity in 10-d cold-shifted leaves was not associated with an increase in alternative oxidase (AOX) protein or AOX1a transcript abundance. By contrast, the amount of uncoupling protein was significantly increased in cold-developed leaves. In conjunction with this, transcript levels of the uncoupling protein-encoding gene UCP1 and the external NAD(P)H dehydrogenase-encoding gene NDB2 exhibited sustained increases following growth in the cold. The data suggest a role for each of these alternative non-phosphorylating bypasses of mitochondrial electron transport at different points in time following exposure to cold, with increased AOP activity being important only in the early stages of cold treatment.

In this study, we investigated whether changes in mitochondrial abundance, ultrastructure and activity are involved in the respiratory cold acclimation response in leaves of the cold-hardy plant Arabidopsis thaliana . Confocal microscopy [using plants with green fluorescence protein (GFP) targeted to the mitochondria] and transmission electron microscopy (TEM) were used to visualize changes in mitochondrial morphology, abundance and ultrastructure. Measurements of respiratory flux in isolated mitochondria and intact leaf tissue were also made. Warm-grown (WG, 25/ 20 ∞ C day/night), 3-week cold-treated (CT) and cold-developed (CD) leaves were sampled. Although CT leaves exhibited some evidence of acclimation (as evidenced by higher rates of respiration at moderate measurement temperatures), it was only the CD leaves that were able to reestablish respiratory flux within the cold. Associated with the recovery of respiratory flux in the CD leaves were: (1) an increase in the total volume of mitochondria per unit volume of tissue in epidermal cells; (2) an increase in the ratio of cristae to matrix within mesophyll cell mitochondria; and (3) an increase in the capacity of the energyproducing cytochrome pathway in mitochondria isolated from whole leaf homogenates. Regardless of growth temperature, we found that contrasting cell types exhibited distinct differences in mitochondrial ultrastructure, morphology and abundance. Collectively, our data demonstrated the diversity and tissue-specific nature of mitochondrial responses that underpin respiratory acclimation to the cold, and revealed the heterogeneity of mitochondrial structure and abundance that exists within leaves.

Using measurements of leaf respiratory O(2) uptake (R), we investigated whether immature and mature Arabidopsis thaliana (ecotype Columbia) leaves differed in their response to temperature. Confocal microscopy (using plants with mitochondrially targeted green fluorescent protein [GFP]) was used to determine whether ontogenetic changes in R are associated with concomitant changes in mitochondrial morphology/abundance. Comparisons were made of warm-grown (25/20°C) leaves, warm-grown leaves shifted to cold (5°C) for 10 days, and cold-developed leaves. Short-term Q(10) values and the ability to cold-acclimate were determined. In warm-grown plants, rates of R per mass were highest in immature leaves, decreasing as leaves developed. Moreover, although mitochondrial size (5.6-6.5 μm(3)) remained constant during development, mitochondrial number per μm(3) declined from 0.01 to 0.003 as leaves expanded (i.e., mitochondrial density decreased). Immature and mature leaves did not differ in Q(10) values but did differ in their ability to cold-acclimate. Whereas mature leaves had clear evidence of cold acclimation (e.g., when measured at 25°C, R was highest in cold-developed leaves), young leaves had none. Collectively, the results highlight the changes in rates of R, mitochondrial density, and biomass allocation associated with leaf development and that changes in respiratory flux associated with acclimation only take place within mature tissues.

Interactions between photosynthetic substrate supply and temperature in determining the rate of three respiration components (leaf, belowground and ecosystem respiration) were investigated within three environmentally controlled, Populus deltoides forest bays at Biosphere 2, Arizona. Over 2 months, the atmospheric CO 2 concentration and air temperature were manipulated to test the following hypotheses: (1) the responses of the three respiration components to changes in the rate of photosynthesis would differ both in speed and magnitude; (2) the temperature sensitivity of leaf and belowground respiration would increase in response to a rise in substrate availability; and, (3) at the ecosystem level, the ratio of respiration to photosynthesis would be conserved despite week-to-week changes in temperature. All three respiration rates responded to the CO 2 concentration-induced changes in photosynthesis. However, the proportional change in the rate of leaf respiration was more than twice that of belowground respiration and, when photosynthesis was reduced, was also more rapid. The results suggest that aboveground respiration plays a key role in the overall response of ecosystem respiration to short-term changes in canopy photosynthesis. The short-term temperature sensitivity of leaf respiration, measured within a single night, was found to be affected more by developmental conditions than photosynthetic substrate availability, as the Q 10 was lower in leaves that developed at high CO 2, irrespective of substrate availability. However, the temperature sensitivity of belowground respiration, calculated between periods of differing air temperature, appeared to be positively correlated with photosynthetic substrate availability. At the ecosystem level, respiration and photosynthesis were positively correlated but the relationship was affected by temperature; for a given rate of daytime photosynthesis, the rate of respiration the following night was greater at 25 than 20 1C. This result suggests that net ecosystem exchange did not acclimate to temperature changes lasting up to 3 weeks. Overall, the results of this study demonstrate that the three respiration terms differ in their dependence on photosynthesis and that, short-and medium-term changes in temperature may affect net carbon storage in terrestrial ecosystems.

We examined the effect of manipulating photosystem II (PSII) redox poise on respiratory flux in leaves of Arabidopsis thaliana. Measurements were made on wild-type (WT) plants and npq4 mutant plants deficient in nonphotochemical quenching (NPQ). Two experiments were carried out. In the first experiment, WT and mutant warmgrown plants were exposed to three different irradiance regimes [75, 150 and 300 mmol photosynthetically active radiation (PAR)], and leaf dark respiration was measured in conjunction with PSII redox poise. In the second experiment, WT and mutant warm-grown plants were shifted to 5°C and 75, 150 or 300 mmol PAR, and dark respiration was measured alongside PSII redox poise in cold-treated and cold-developed leaves. Despite significant differences in PSII redox poise between genotypes and irradiance treatments, neither genotype nor growth irradiance had any effect upon the rate of respiration in warm-grown, coldtreated or cold-developed leaves. We conclude that changes in PSII redox poise, at least within the range experienced here, have no direct impacts on rates of leaf dark respiration, and that the respiratory cold acclimation response is unrelated to changes in chloroplast redox poise.